Kir3.1 and Kir3.2A heterotetramers form the cloned counterpart of the neuronal G-protein-gated inwardly rectifying K⁺ channel. The channel is activated by the release of Gβγ from heterotrimeric G-proteins of the Gio class after activation by agonist of a G-protein-coupled receptor. In order to understand the dynamics of this process better we have examined the ability of receptor- G-protein fusions (A1 adenosine receptor fused to Giα1, α2A adrenergic receptor fused to Giα1) to activate current using the whole-cell configuration of the patch-clamp technique. Currents were recorded in 140 mM extracellular KCl-based solution. The fused Gio α subunits contain mutations (C-I for α2A or C-G for A1) of the most C-terminal cysteine rendering them resistant to the actions of pertussis toxin (PTx). We transiently transfected these constructs into a stable HEK293 cell line expressing Kir3.1 and Kir3.2A. In PTx-treated cells (100 ng ml^-1 for 16 h) both fusions were able to significantly activate currents (pA pF^-1) after application of the appropriate agonist (α2A-Giα1-C-I basal: -31.6 ± 4.5, +10 µM noradrenaline: -151.4 ± 24.1 (n = 22); A1-Giα1-C-G basal: -22.4 ± 4.4, +1 µM 5Ì-N-ethylcarboxyamidoadenosine (NECA, an adenosine receptor agonist): -166.7 ± 36.1 (n = 18)).

We investigated this further for the A1-Giα1C-G fusion construct by developing stable lines co-expressing channel and receptor construct. Using a fast perfusion system, we have examined the dynamics of current activation and deactivation following 2 and 20 s agonist applications of 1 µM NECA at a holding potential of -60 mV in two clonal isolates in each condition. Data were analysed using one-way ANOVA with Dunnett’s post-hoc test (n.s., not significant; *P < 0.05, ***P < 0.001) and presented as means ± S.E.M. Basal currents (pA pF^-1) were smaller in the fused lines but this difference was not significant. NECA-activated peak currents (pA pF^-1) were similar in both conditions (Fused Clone1: -196 ± 40, n = 12; Fused Clone2: -334 ± 84, n = 11; A1 Clone1: -189 ± 31, n = 7; A1 Clone2: -227 ± 43, n = 7, all n.s.). In addition, we have determined receptor expression (fmol (µg protein)^-1) using radioligand binding of the A1 receptor antagonist, [³H] DPCPX (8 nM) (Fused Clone1: 73 ± 13, n = 9; Fused Clone2: 59 ± 20, n = 5; A1 Clone1: 15 ± 2, n = 5, *; A1 Clone2: 29 ± 7, n = 6, *). Activation kinetics were characterised by an initial lag phase and subsequent rising phase and were corrected for system dead time by using Ba²⁺ block. The fusion of receptor to G-protein resulted in a statistically significant slowing of activation and increased rate of deactivation in the fused receptor cell lines, despite increased receptor expression (see Table 1). These data support the concept of a catalytic mechanism for G-protein activation in which receptor to G-protein diffusion is not rate limiting and an unlinked receptor is able to activate multiple G-proteins.

This work was supported by The Wellcome Trust and Royal Society.